Terpenes or terpenoids are a structurally diverse and a very large group of organic compounds commonly found in plants ranging from essential and universal primary metabolites such as sterols, carotenoids and hormones to more complex and unique secondary metabolites. Terpenes are hydrocarbons assembled of five carbon terpene or isoprene subunits providing the carbon skeleton. Terpenoids are modified terpenes which typically comprise also oxygen; however, terpenes and terpenoids are often used interchangeably. Terpenoids are classified accordingly to the length of the isoprene units as for example hemiterpenoids consisting of one, monoterpenoids consisting of two, sesquiterpenoids consisting of three and diterpenoids consisting of four isoprene units.
The early core steps in terpenoid biosynthesis are well characterised in both eukaryotes and prokaryotes. The primary building blocks are isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are used for the synthesis of the compounds geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP).
Geranylgeranyl diphosphate (GGPP) is the precursor for the synthesis of a variety of diterpenes, which first steps are catalysed by diterpene synthases. For the conversion of terpenes to terpenoids, a number of enzymes may be involved. Cytochrome P450s are typically required for the addition of oxygen, whereas BAND acyltransferase are often involved in the addition of acyl groups. The enzymes involved in the biosynthesis of some diterpenes, for example paclitaxel, have been partially characterised, with sequences for a diterpene synthase (Wildung & Croteau, 1996. J. Biol. Chem. 271: 9201-9204) a number of cytochrome P450s (Schoendorf et al., 2001. PNAS 98: 1501-1506; Jennewein et al. 2001: PNAS 98: 13595-13600; Jennewein et al. 2003. Arch. Biochem. Biophys. 413: 262-270; Jennewein et al., 2004. Chem. Biol. 11: 379-387; Chau et al., 2004a: Chem. Biol. 11: 663-672; Chau et al. 2004b. Arch. Biochem. Biophys. 427: 48-57) and a number of acyltransferases (Walker & Croteau, 2000. PNAS 97: 583-587; Walker et al., 2000. Arch. Biochem. Biophys. 274: 371-380; Chau et al 2004c. Arch. Biochem. Biophys. 430: 237-246) being described. However, the enzymes involved in the synthesis of the vast majority of diterpenoids produced by other plant species remain unknown.
Diterpenes form the basis for many biologically important compounds such as retinol, retinal, and phytol and some compounds have shown antimicrobial and anti-inflammatory properties. A large number of diterpenes have been isolated from plants belonging to the family of Euphorbiaceae. The Euphorbiaceae or spurge family is a large family of flowering plants found all over the world, with some synthesising compounds of considerable biological activity such as ingenol mebutate (Euphorbia peplus), resiniferatoxin (E. resinifera), prostratin (E. cornigera), jatrophanes (Jatropha sp.) and jatrophone (Jatropha sp.).
Although the beneficial effects of some diterpenes are known such as ingenol mebutate which is licensed to treat actinic keratosis, or resiniferatoxin which is currently being tested in Phase II clinical trials for its analgesic effects, sufficient supply is still hampered by the lack of or inefficient chemical synthesis. Similarly, extraction of active compounds from the plant biomass is a complex process requiring several steps and various solvents, and moreover the yield is typically very low. Methods and processes enabling extraction of diterpenes from plants are disclosed in U.S. Pat. Nos. 4,361,697 and 6,228,996.
Bacteria and yeast have been successfully used to engineer biosynthetic pathways for the production of some desired chemical compounds from inexpensive carbon sources. The terpenoid artemisinic acid, a precursor for the anti-malaria drug artemesinin, has been successfully synthesised in yeast using this approach. Bacterial or yeast expression systems are often advantageous over other expression systems as they are easily maintained and various methods are available allowing straightforward expression of transgenic genes. The biosynthesis of isoprenoids using a genetically modified bacterial host cell comprising one or more enzymes of the mevalonate pathway is disclosed in patent application WO2008/039499.
The applicants of the present application have identified a number of cytochrome P450 encoding genes involved in the biosynthesis of diterpenoids.